Small-Animal PET Study of Adenosine A1 Receptors in Rat Brain ...

Small-Animal PET Study of Adenosine A1 Receptors in Rat Brain: Blocking Receptors and Raising Extracellular Adenosine Soumen Paul1, Shivashankar Khanapur1, Anna A. Rybczynska1, Chantal Kwizera1, Jurgen W.A. Sijbesma1, Kiichi Ishiwata2, Antoon T.M. Willemsen1, Philip H. Elsinga1,3, Rudi A.J.O. Dierckx1,3, and Aren van Waarde1 1Nuclear Medicine and Molecular Imaging, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands; 2Positron Medical Center, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan; and 3Department of Nuclear Medicine, University Hospital Ghent, Ghent, Belgium

Activation of adenosine A1 receptors (A1R) in the brain causes sedation, reduces anxiety, inhibits seizures, and promotes neuroprotection. Cerebral A 1 R can be visualized using 8dicyclopropylmethyl-1-11C-methyl-3-propyl-xanthine (11C-MPDX) and PET. This study aims to test whether 11C-MPDX can be used for quantitative studies of cerebral A1R in rodents. Methods: 11CMPDX was injected (intravenously) into isoflurane-anesthetized male Wistar rats (300 g). A dynamic scan of the central nervous system was obtained, using a small-animal PET camera. A cannula in a femoral artery was used for blood sampling. Three groups of animals were studied: group 1, controls (saline-treated); group 2, animals pretreated with the A1R antagonist 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX, 1 mg, intraperitoneally); and group 3, animals pretreated (intraperitoneally) with a 20% solution of ethanol in saline (2 mL) plus the adenosine kinase inhibitor 4amino-5-(3-bromophenyl)-7-(6-morpholino-pyridin-3-yl)pyrido [2,3-d] pyrimidine dihydrochloride (ABT-702) (1 mg). DPCPX is known to occupy cerebral A1R, whereas ethanol and ABT-702 increase extracellular adenosine. Results: In groups 1 and 3, the brain was clearly visualized. High uptake of 11C-MPDX was noted in striatum, hippocampus, and cerebellum. In group 2, tracer uptake was strongly suppressed and regional differences were abolished. The treatment of group 3 resulted in an unexpected 40%–45% increase of the cerebral uptake of radioactivity as indicated by increases of PET standardized uptake value, distribution volume from Logan plot, nondisplaceable binding potential from 2tissue-compartment model fit, and standardized uptake value from a biodistribution study performed after the PET scan. The partition coefficient of the tracer (K1/k2 from the model fit) was not altered under the study conditions. Conclusion: 11C-MPDX shows a regional distribution in rat brain consistent with binding to A1R. Tracer binding is blocked by the selective A1R antagonist DPCPX. Pretreatment of animals with ethanol and adenosine kinase inhibitor increases 11C-MPDX uptake. This increase may reflect an increased availability of A1R after acute exposure to ethanol.

Received Jan. 17, 2011; revision accepted Apr. 11, 2011. For correspondence or reprints contact: Aren van Waarde, Nuclear Medicine and Molecular Imaging, University Medical Center Groningen, University of Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands. E-mail: [email protected] COPYRIGHT ª 2011 by the Society of Nuclear Medicine, Inc.

Key Words: receptors; adenosine A1; adenosine kinase inhibitor; brain; positron emission tomography (PET); ethanol J Nucl Med 2011; 52:1293–1300 DOI: 10.2967/jnumed.111.088005

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he adenosine receptor (R) family consists of the A1, A2A, A2B, and A3 subtypes. A1 and A3R inhibit, whereas A2A and A2B stimulate, production of the second messenger, 39,59-cyclic adenosine monophosphate. A1R and A2AR are activated by nanomolar concentrations of adenosine, whereas A2B and A3R become activated only when adenosine levels rise into the micromolar range because of inflammation, hypoxia, or ischemia (1–3). A1Rs are highly expressed and extensively distributed in various regions of the human brain such as the hippocampus, cerebral cortex, thalamic nuclei, and basal ganglia (4,5). In the central nervous system, adenosine acts as an endogenous modulator of neurotransmission (6), a neuroprotectant (7), and an anticonvulsant (8). Its neuroprotective action is mediated via A1R and may be associated with inhibition of the release of excitatory neurotransmitters, hyperpolarization of neurons, and inhibition of Ca21 channels (9). Adenosine acts also as an analgesic, by affecting nociceptive afferent and transmission neurons via A1R (10). A1R agonists usually stimulate (11), whereas A1R antagonists diminish, sleep (12). Thus, such compounds may be therapeutically useful. Yet, A1R agonists have failed to undergo successful clinical development because of dose-limiting cardiovascular side effects. Adenosine kinase inhibitors (AKIs) represent an alternative treatment strategy. Adenosine kinase (AK) catalyzes a phosphorylation reaction, converting adenosine to adenosine monophosphate (13,14). The inhibition of AK decreases the cellular reuptake of adenosine, resulting in increased local adenosine concentrations (14). The feasibility of raising adenosine availability in the central nervous system by inhibiting AK has been demonstrated in hippocampal and spinal cord slices (15) and by in vivo studies on extracel-

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lular adenosine in rat striatum, which was increased up to 10-fold (16). The psychoactive drug ethanol also raises extracellular levels of adenosine in the brain (up to 4-fold (17)) by augmenting the rate of adenosine formation (18) and inhibiting adenosine uptake via nucleoside transporters (18–20). The anxiolytic, sedating, and motor-impairing effects of ethanol are related to its interaction with adenosinergic signaling. PET with a radiolabeled A1R ligand may allow study of the involvement of A1R in the pathophysiology of disease, the response of the A1R population to therapy, and assessment of the occupancy of A1R by therapeutic drugs. Several positronemitting A1R ligands have been prepared for this purpose, but only 2 have been widely used: 8-dicyclopropylmethyl1-11C-methyl-3-propylxanthine (11C-MPDX) (21) and 18F-8cyclopentyl-3-(3-fluoropropyl)-1-propylxanthine (5). Both ligands bind with high affinity and selectivity to A1R in vivo (Ki and Kd values, 3.0 and 4.4 nM, respectively). Because small-animal PET studies with 11C-MPDX had not been performed previously, we tested this ligand for quantitative small-animal PET studies in rodents with the intention of later using this technique for the assessment of changes of A1R density in rodent models of human disease. In addition, we examined the impact of raised levels of extracellular adenosine on the cerebral binding of 11C-MPDX. MATERIALS AND METHODS Chemicals Ethanol and triethylamine were purchased from Merck. The adenosine A1 antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) was a product of Sigma, and the potent nonnucleoside 4-amino-5-(3-bromophenyl)-7-(6-morpholino-pyridin-3-yl)pyrido [2,3-d]pyrimidine dihydrochloride (ABT-702) was obtained from Tocris. Stock solutions of DPCPX and ABT-702 were prepared in dimethyl sulfoxide. The radioligand 11C-MPDX was prepared by reaction of 11C-methyl iodide with the appropriate 1-N-desmethyl precursor. Briefly, 11C-methyl iodide was trapped in 0.3 mL of N,Ndimethylformamide containing 1 mg of 1-N-desmethyl precursor and 5 mL of NaOH and was heated at 120C for 5 min. After 1.0 mL of 0.1 M HCl had been added, the solution was loaded onto a high-performance liquid chromatography column (Econosphere, C18, 5 mm [Altech]; 10 · 250 mm) and eluted with a mixture of 0.1 M NaH2PO4 and ethanol (70/30) at a flow rate of 4 mL/min. The fractions containing 11C-MPDX were collected. Retention time of 11C-MPDX was 14 min. The decay-corrected radiochemical

yield was 35% 6 5% (based on 11C-methyl iodide), the specific radioactivity was greater than 11 TBq/mmol at the moment of injection, and the radiochemical purity was greater than 98%. Animal Model The animal experiments were performed by licensed investigators in compliance with the Law on Animal Experiments of The Netherlands. The protocol was approved by the Committee on Animal Ethics of the University of Groningen. Male Wistar rats were maintained at a 12-h light/12-h dark regime and were fed standard laboratory chow ad libitum (body weights are provided in Table 1). Small-Animal PET Scanning In most experiments, 2 rats were scanned simultaneously, using a Focus 220 microPET camera (Siemens-Concorde). Animals were anesthetized with a mixture of isoflurane/air (inhalation anesthesia, 5% ratio during induction, later reduced to ,2%). A cannula was placed in a femoral artery for blood sampling. Rats were under anesthesia for 30–40 min before tracer injection (time required for cannulation and transmission scan). The tracer (11CMPDX) was injected through the penile vein (injected dose is given in Table 1). A list-mode protocol was used (76 min, brain in the field of view). Scanning was started during injection of radioactivity in the lower rat; the upper animal was injected 16 min later. The animal that was injected last was also anesthetized at a later moment. Thus, the duration of anesthesia was similar in all study groups. A series of blood samples (14 samples; volume, 0.10–0.15 mL) was drawn, initially in rapid succession (every 15 s) and later at longer intervals (#30 min). Plasma was acquired from these samples by short centrifugation (Eppendorf centrifuge, 5 min at 13,000 rpm). Radioactivity in 25 mL of plasma was counted and used as an arterial input function. For examination of the specificity of tracer binding, 5 animals were pretreated by intraperitoneal injection of DPCPX (1 mg, in 0.3 mL of dimethyl sulfoxide, 15–20 min before injection of the tracer). For examination of the impact of raised levels of extracellular adenosine on 11C-MPDX binding, 5 other rats received ethanol (2 mL of a 20% solution in saline intraperitoneally) and the AKI ABT-702 (1 mg, in 0.3 mL of dimethyl sulfoxide intraperitoneally). Both ethanol and ABT-702 were administered 15– 20 min before injection of 11C-MPDX. Control animals (n 5 5) received saline only. The ethanol dose that we administered corresponds to substantial consumption of alcohol in humans (about six 0.33-L bottles of normal beer containing 5% alcohol). List-mode data were reframed into a dynamic sequence of 8 · 30, 3 · 60, 2 · 120, 2 · 180, 3 · 300, 1 · 480, 2 · 600, and 1 · 960 s frames. The data were reconstructed per time frame using an iterative reconstruction algorithm (attenuation-weighted 2-dimensional

TABLE 1 Animal Data Injection in. . . Group

Body weight (g)

Control (n 5 5) DPCPX (n 5 5) Ethanol and ABT-702 (n 5 5) Metabolite analysis (n 5 6)

299 314 302 314

6 6 6 6

8 18 16 14

MBq 24 26 34 20

6 6 6 6

10 14 11 12

Data are mean 6 SD.

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nmol

ROI size (cm3)

6 6 6 6

1.02 6 0.02 1.02 6 0.04 1.02 6 0.03 —

2.2 2.4 3.1 1.8

0.9 1.3 1.0 1.1

ordered-subset expectation maximization, provided by Siemens; 4 iterations, 16 subsets; zoom factor, 2). The final datasets consisted of 95 slices, with a slice thickness of 0.8 mm and an inplane image matrix of 128 · 128 pixels of size 1 · 1 mm. Datasets were fully corrected for random coincidences, scatter, and attenuation. A separate transmission scan (duration, 515 s) was acquired for attenuation correction. That scan was made before the emission scan. Images were smoothed with a gaussian filter (1.35 mm in both directions). Small-Animal PET Data Analysis Three-dimensional regions of interest (ROIs) were manually drawn around the entire brain. Time–activity curves and volumes (cm3) for the ROIs were calculated, using standard software (AsiPro, version 6.2.5.0; Siemens-Concorde). PET standardized uptake values (SUVs) for brain radioactivity were calculated, using measured body weights and injected doses and assuming a specific gravity of 1 g/mL for brain tissue and blood plasma. Dynamic PET data were analyzed using plasma radioactivity from arterial blood samples as an input function and a graphical method according to Logan (22). Because 11C-MPDX proved to be rapidly cleared but slowly metabolized, no metabolite correction of the input function was performed. The error introduced by this procedure (overestimation of the true plasma input) is 10.0% and identical in all study groups; thus, we concluded that metabolite correction could be omitted. Software routines for MatLab 7

(The MathWorks), written by Dr. Antoon T.M. Willemsen (University Medical Center Groningen), were used for curve fitting. The Logan fit was started at 10 min. The cerebral distribution volume (VT) of the tracer was estimated from the Logan plot. The dynamic PET data were also analyzed using the same input function and software routines, a 2-tissue-compartment model (2TCM), and a fixed blood volume of 3.6%. The partition coefficient (K1/k2) and nondisplaceable binding potential (BPND) (k3/k4) of 11C-MPDX were estimated from the model fit. Similar methods were used previously by Kimura et al. (23) for quantification of A1R in the human brain. However, A1Rs are significantly expressed in rat cerebellum, in contrast to human cerebellum, in which A1R density is negligible. Therefore, the cerebellum cannot be used as a reference region in small-animal PET studies of the rodent brain. Biodistribution Studies After the scanning period, the anesthetized animals were sacrificed. Blood was collected, and plasma and a cell fraction were obtained from the blood sample by short centrifugation (5 min at 1,000g). Several brain areas and peripheral tissues (Table 2) were excised. All tissue samples were weighed. The radioactivity in tissue samples was measured using a g-counter, applying a decay correction. The results were expressed as dimensionless SUVs. The parameter SUV is defined as tissue activity concentration (MBq/g) · body weight (g)/injected dose (MBq).

TABLE 2 Biodistribution Data of Tissue

Control animals (n 5 5)

Amygdala Olfactory bulb Cerebellum Cingulate Entorhinal Frontal Hippocampus Medulla Parietal, temporal, and occipital cortex Pons Striatum Bone Colon Duodenum Fat Heart Ileum Kidney Liver Lung Muscle Pancreas Plasma Red cells Spleen Trachea

11C-MPDX,

80 Minutes After Injection

DPCPX-pretreated (n 5 5)

Difference vs. control

Ethanol and ABT-702– treated (n 5 5)

Difference vs. control

0.75 0.64 1.34 0.88 0.89 0.89 1.09 0.75 0.97

6 6 6 6 6 6 6 6 6

0.09 0.18 0.29 0.16 0.10 0.20 0.14 0.18 0.20

0.27 0.34 0.46 0.35 0.37 0.30 0.31 0.42 0.34

6 6 6 6 6 6 6 6 6

0.05 0.07 0.09 0.08 0.04 0.08 0.08 0.11 0.07

,0.0001 ,0.01 0.0001 0.0001 ,0.0001 0.0002 ,0.0001 0.01 0.0001

1.12 0.75 2.16 1.13 1.27 1.16 1.47 1.51 1.40

6 6 6 6 6 6 6 6 6

0.12 0.27 0.42 0.23 0.25 0.26 0.24 0.29 0.48

,0.0005 NS ,0.01 0.06 ,0.01 NS 0.01 ,0.001 NS

0.89 1.01 0.24 0.83 1.12 1.95 0.69 1.52 1.09 2.94 0.70 0.40 0.98 0.77 0.34 0.95 0.73

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

0.21 0.13 0.04 0.21 0.33 1.15 0.14 0.52 0.18 0.40 0.09 0.09 0.18 0.06 0.04 0.14 0.15

0.44 0.31 0.22 0.61 0.95 1.52 0.66 1.19 1.26 4.43 0.73 0.48 1.01 0.73 0.38 0.65 0.81

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

0.14 0.07 0.09 0.19 0.57 0.19 0.08 0.41 0.07 1.03 0.07 0.07 0.23 0.13 0.09 0.06 0.31

,0.01 ,0.0001 NS NS NS NS NS NS 0.08 ,0.02 NS NS NS NS NS ,0.005 NS

1.45 1.18 0.24 0.71 1.19 1.06 0.85 1.38 1.50 3.63 0.88 0.53 1.48 0.71 0.44 1.06 0.85

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

0.30 0.31 0.09 0.06 0.17 0.24 0.17 0.43 0.50 0.68 0.07 0.07 0.22 0.13 0.07 0.26 0.23

,0.01 NS NS NS NS NS NS NS NS NS ,0.01 ,0.05 ,0.005 NS ,0.02 NS NS

SUVs (mean 6 SD) are listed. NS 5 not significant.


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Metabolite Analysis A separate group of animals (n 5 6) was used for metabolite analysis. In these rats, a small-animal PET scan was obtained and a biodistribution study was performed, but a smaller series of arterial blood samples was drawn (at intervals of 5, 10, 20, 40, and 60 min after tracer injection, volume increasing from 0.3 to 0.7 mL). Plasma was acquired by short centrifugation (Eppendorf centrifuge, 5 min at 13,000 rpm). Protein was removed by mixing plasma with an equivalent volume of 20% trichloroacetic acid in acetonitrile, followed again by short centrifugation. The proteinfree supernatant was injected into a high-performance liquid chromatography system (stationary phase, mBondapak, 7.8 · 300 mm [Waters]; mobile phase, 3% triethylamine and phosphate, pH 2.0: acetonitrile, 60:40 v/v, with a flow rate of 2 mL/min). The retention time of authentic 11C-MPDX (and nonradioactive MPDX) was about 11 min. Two radioactive metabolites eluted at shorter retention times (6 and 8 min, respectively). This reversed-phase system is a slightly modified version of a published analytic procedure (24). One-milliliter samples of the eluate were collected at 0.5-min intervals. Radioactivity in these samples was determined with a g-counter and was automatically corrected for decay.

RESULTS

uptake of the tracer rapidly increased to a maximum, which was already reached between 7 and 12 min, and was followed by washout. In animals pretreated with DPCPX, only a rapid washout of tracer was observed, and the cerebral uptake of 11C was strongly reduced. In rats pretreated with ABT-702 and ethanol, cerebral uptake of radioactivity was significantly increased, compared with the control group. Maximal tracer uptake now occurred after 13–20 min and was followed by washout. On the basis of SUVs measured with small-animal PET, A1R densities reported in the literature (e.g., 5,003 fmol of protein per milligram in rat hippocampus (25)), injected masses of the tracer (Table 1), and assuming that cerebral tissue contains 10% protein, we estimate that less than 5% of the A1R population in the rat brain was occupied by 11CMPDX under the conditions of our study. Kinetics of radioactivity in rat plasma after injection of 11C-MPDX are presented in Figure 2. A rapid, biexponential clearance was observed in all groups. Treatment of animals with DPCPX or a combination of ethanol and AKI (ABT-702) did not significantly affect tracer clearance from the plasma compartment. Areas under the curve (percentage of control) were 100.0 6 8.4, 95.5 6 6.6, and 96.0 6 7.5 for the baseline, ethanol and ABT-702, and DPCPX groups, respectively (mean 6 SEM).

Small-Animal PET Images

Metabolite Analysis

Small-animal PET images acquired after injection of 11C-MPDX are presented in Figure 1. In saline-treated control animals, the brain was clearly visualized. High tracer uptake was observed in the hippocampus, cerebellum, and striatum (left panel) in addition to some areas of the cortex (image not shown). After pretreatment of rats with DPCPX, cerebral uptake of the tracer was strongly reduced, and regional differences in tracer uptake were no longer apparent (middle panel). When animals were pretreated with ethanol and the AKI ABT-702, a global increase of tracer uptake was noted, compared with the control group (right panel).

The appearance of radiolabeled metabolites in rat plasma after injection of 11C-MPDX was studied in 2 untreated control animals, 2 rats treated with ethanol and ABT-702, and 2 rats pretreated with DPCPX. Injected 11C-MPDX was found to be hardly metabolized. The fraction of parent compound decreased from almost 100% at time zero to 82%– 84% at 60 min (Fig. 3). Pretreatment did not affect the rate of tracer metabolism.

Statistical Tests Differences between groups were analyzed using 1-way ANOVA. A probability smaller than 0.05 was considered statistically significant.

Kinetics of Radioactivity in Brain and Plasma

Cerebral kinetics of 11C-MPDX–derived radioactivity (PET SUV in the whole brain as a function of time) are presented in Figure 2. In saline-treated control animals,

Biodistribution Data

Biodistribution data of 11C, acquired 80 min after injection of 11C-MPDX, are presented in Table 2. Pretreatment of animals with DPCPX resulted in a highly significant reduction of tracer uptake in all studied brain areas. Among peripheral organs, a significant reduction of tracer uptake was observed only in the spleen. DPCPX treatment caused

FIGURE 1. Small-animal PET images of rat brain acquired after injection of 11CMPDX (summed frames from 2 min to end of scan, SUV maximum set to 3): untreated control animal (left), animal pretreated with DPCPX (middle), and animal pretreated with ethanol and ABT-702 (right).

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Treatment of rats with ethanol and ABT-702 increased uptake of radioactivity in the brain. This increase was statistically significant in the amygdala, cerebellum, entorhinal cortex, hippocampus, medulla, and pons. In other brain areas, an increase was also noted but this trend did not reach statistical significance because of a relatively large individual variance in the study groups. Outside the brain, increases of tracer uptake were noted in lungs, skeletal muscle, pancreas, and red blood cells after treatment of animals with ethanol and ABT-702. Graphical Analysis of PET Data

VT of tracer was calculated using a Logan plot (Fig. 4), time–activity curves from an ROI drawn around the entire brain, and radioactivity counts from arterial blood samples. VT of tracer was significantly decreased (by 63%) after pretreatment of animals with DPCPX and significantly increased (by 39%) after pretreatment with ethanol and ABT-702 (Table 3). Compartment Modeling of PET Data

FIGURE 2. Kinetics of 11C-MPDX–derived radioactivity in rat brain (A) and plasma (B). Error bars indicate SEM. Plasma data are not corrected for metabolites. 5 control group; s 5 ethanol and ABT-702–treated animals; , 5 animals pretreated with DPCPX.

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a significant increase of the amount of radioactivity in the liver. Renal uptake of the tracer appeared to be increased as well, but the change was relatively small and this trend did not reach statistical significance.

A 2TCM was fitted to time–activity curves from an ROI drawn around the entire brain, using radioactivity counts from arterial blood samples as an input function. A 1-tissuecompartment model could not be fitted to the time–activity curves of control and ethanol and ABT-702–treated animals at all, in contrast to a 2TCM. Thus, the 2TCM was clearly superior. The partition coefficient of 11C-MPDX (ratio K1/k2 from the model fit) was not significantly affected by any of the treatments, in contrast to BPND (Table 3). BPND was reduced to zero after treatment of animals with DPCPX and significantly increased (by 54%) after treatment with ethanol and ABT-702. VT calculated from the 2TCM fit corresponded closely to VT acquired by graphical (Logan) analysis of the PET data, although the intraindividual variability was greater. VT (from the model fit) was significantly reduced (by 63%) after pretreatment of animals with DPCPX and significantly increased (by 42%) after treatment with ethanol and ABT-702. DISCUSSION Specificity of

FIGURE 3. Fraction of plasma radioactivity representing parent 11C-MPDX. Pooled data are used because group differences were not observed. Error bars indicate SEM.

11C-MPDX

Binding

The regional distribution of radioactivity in the rat brain after injection of 11C-MPDX (Fig. 1, left) suggests that this tracer is capable of visualizing regional A1R densities. Further evidence for specific in vivo binding of 11C-MPDX was obtained by pretreating animals with the subtype-selective antagonist DPCPX. In pretreated animals, the brain uptake of radioactivity after injection of 11C-MPDX was strongly suppressed, and regional differences were no longer evident (Fig. 1, middle; Fig. 2, top). A biodistribution study, performed at 80 min after tracer injection, confirmed that uptake of radioactivity was reduced by DPCPX to a low value that was homogeneous throughout the brain (Table 2). The greatest declines were observed in the hippocampus (72%), striatum (69%), cerebellum (66%), frontal cortex (66%), parietal cortex (65%), and amygdala (64%). Outside the brain, a


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TABLE 3 Results from Graphical Analysis and Compartment Modeling of PET Data (ROI Drawn Around Entire Brain) Parameter Control rats DPCPX-pretreated Ethanol and ABT-702–pretreated

VT (Logan)

K1/k2 (2TCM)

BPND (k3/k4) (2TCM)

VT (2TCM)

1.52 6 0.18 0.57 6 0.05 P , 0.0001 2.12 6 0.25 P , 0.005

0.62 6 0.17 0.58 6 0.07 P 5 NS 0.68 6 0.17 P 5 NS

1.52 6 0.10 0.00 P , 0.0001 2.34 6 0.64 P , 0.05

1.56 6 0.37 0.58 6 0.07 P , 0.0001 2.21 6 0.44 P , 0.05

Mean 6 SD P values relate to effect of pretreatment, compared with untreated controls. NS 5 not significant.

reduction of 11C-MPDX uptake was observed only in the spleen, possibly reflecting specific binding of the tracer to A1R, because A1Rs are involved in splenic contraction (26). DPCPX caused a significant increase of the levels of radioactivity in rat liver and tended to increase renal activity levels as well (Table 2), indicating that after blocking of the receptor compartment, a greater fraction of the injected dose is taken up by organs involved in tracer excretion. To further test the origin of the PET signal, we plotted the specific binding of 11C-MPDX in various brain areas (uptake in saline-treated control animals minus uptake in animals pretreated with DPCPX, Table 2) against regional A1R numbers, known from autoradiography (27–29). The regions plotted were the amygdala, cerebellum, cingulate, entorhinal, and frontal cortices; hippocampus; medulla; parietal– temporal–occipital cortex; pons; and striatum. Receptor density in the hippocampus (main target region) was set to 100%, to allow the use of data from several published studies. An excellent correlation was observed between literature values for A1R density and 11C-MPDX uptake at 80 min after injection (Fig. 5). Data analysis of the cerebral time–activity curves in saline- and DPCPX-treated animals also confirmed specific binding of 11C-MPDX to cerebral A1R. Tracer VT in the entire brain—calculated either by graphical analysis or by kinetic modeling of the PET data—showed a decline (.60%) similar to that of tracer SUV measured after 80 min (Table 3). In contrast, BPND of 11C-MPDX estimated by fitting a 2TCM was reduced to zero after pretreatment of animals with DPCPX (Table 3), suggesting that specific binding of 11C-MPDX is absent in DPCPX-treated animals.

transporters in cellular membranes, particularly the type 1 equilibrative nucleoside transporter (19,20,30,31). Increased binding of adenosine to cerebral A1R is believed to be an important factor underlying the motor incoordination (32–35) and sleep-promoting (36) effects of ethanol.

Effect of Ethanol and Inhibition of Adenosine Kinase

Acute administration of ethanol is known to result in strong increases of extracellular adenosine both in cell culture and in the rat brain in vivo, which can be assessed by microdialysis (17). Two different mechanisms may underlie this effect of ethanol. First, ethanol is metabolized to acetate and acetyl-coA before entering the tricarboxylic acid cycle. Increased flux through acetyl coA-synthetase leads to increased production of adenosine from adenosine monophosphate via 59-nucleotidase and stimulation of cellular adenosine release (18). Second, ethanol blocks nucleoside

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FIGURE 4. Logan plots (A) of control, ethanol and ABT-702–treated, and DPCPX-treated rats and 2TCM fit (B) for animal pretreated with ethanol and ABT-702. (A) 5 control group; s 5 ethanol and ABT702–treated animals; n 5 animals pretreated with DPCPX. (B) n 5 measured activity in brain; h 5 fitted activity in brain, ♢ 5 specific binding in brain; , 5 nondisplaceable binding in brain and tracer in plasma; ♦ 5 tracer in plasma.

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The coadministration of an AKI (e.g., ABT-702) with ethanol leads to even stronger increases of extracellular adenosine, because phosphorylation of adenosine by the enzyme AK is normally the primary route of adenosine metabolism. Inhibition of AK decreases the rate of adenosine inactivation and locally enhances extracellular adenosine concentrations—not at baseline but rather under conditions of increased formation of adenosine. Orally administered AKIs are known to raise regional concentrations of endogenous adenosine in the brain (15,16), and for this reason, these compounds have therapeutic potential as analgesic, antiinflammatory, and antiepileptic agents (14,37). Because both ethanol administration and AK inhibition are known to increase the levels of extracellular adenosine, we expected to observe a decreased binding of the PET tracer 11C-MPDX in the rat brain after acute treatment of rats with ethanol and ABT-702. Competition of adenosine for tracer binding to A1R is likely to occur, because the affinities of adenosine and 11C-MPDX for cerebral A1R are in the same (nanomolar) range (38). However, in ethanol and ABT-702–pretreated animals, we observed a paradoxic increase rather than a decrease of cerebral tracer binding. Statistically significant increases occurred in PET SUV (Figs. 1 and 2), SUV from an ex vivo biodistribution study (Table 2), and tracer VT (Table 3). Kinetic modeling was performed to gain more insight into the mechanisms underlying the paradoxic increase of cerebral radioactivity induced by ethanol and ABT-702. Fitting of a 2TCM to the cerebral time–activity curves of control and treated animals indicated that the partition coefficient of the tracer (K1/k2) was not affected by treatment, in contrast to BPND, which showed a significant increase (Table 3). The fit data, and also our data on tracer clearance (Fig. 2) and metabolism (Fig. 3), suggest that tracer delivery is not changed by treatment. Increases of the apparent A1R densities (on average, 35%; maximally, 55%) in the brain of rats and mice have been reported, both after acute (32,39) and after chronic (39,40) administration of ethanol, using ex vivo binding assays. Such changes could cause increased cerebral binding of 11C-MPDX after treatment of rodents with ethanol and ABT-702. However, further studies are necessary to identify the mechanism underlying increased binding of the A1R ligand under these conditions. CONCLUSION

Our data suggest that regional A1R densities in rat brain can be assessed using the tracer 11C-MPDX and small-animal PET. In the brain of untreated control animals, the highest levels of tracer uptake were observed in target regions with a high density of A1R, such as the hippocampus, striatum, cerebellum, and cerebral cortex (Fig. 1). Pretreatment of animals with the specific A1R antagonist DPCPX resulted in a strong suppression of tracer uptake in the central nervous system and an abolishment of the regional differences (Fig. 1; Table 2). Specific binding of the tracer in various brain regions corresponded closely to regional A 1R densities

FIGURE 5. Correlation between specific in vivo binding of 11CMPDX and regional A1R density as known from autoradiography.

known from autoradiography (Fig. 5). Tracer binding can be quantified both by graphical analysis (Logan plot, calculation of VT) and by kinetic modeling (BPND or VT from 2TCM, Table 3). The PET data did not provide evidence for increased competition of endogenous adenosine after acute treatment of animals with ethanol and ABT-702, but a globally increased binding of 11C-MPDX was noted in the rat brain (Figs. 1 and 2; Tables 2 and 3), which may correspond to increases of apparent A1R density reported in the literature. Further studies are necessary to elucidate the mechanisms underlying the enhanced 11C-MPDX binding after treatment of animals with ethanol and ABT-702. DISCLOSURE STATEMENT

The costs of publication of this article were defrayed in part by the payment of page charges. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734. ACKNOWLEDGMENT

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